Proinflammatory cytokines, such as TNF IL-1, IL-8, are produced by leukocytes in response to bacteria or their components. The inflammatory response involves leukocyte transcriptional activation and specific gene expression. Most studies have investigated the activation of transcription factors, such as NF-kappaB, following exposure to a single inducer but, in vivo, multiple pathogenic inducers exist simultaneously at sites of infection and inflammation. In the last funding period, we have found that bacterial lipopolysaccharide (LPS) and a formylated peptide (fMLP), each induce activation of cytokine gene transcription in leukocytes. Interestingly, fMLP induces some intracellular signaling events that differ from those induced by LPS. Both stimuli, however, activate the signaling pathways that converge to IkB phosphorylation and NF-kappaB activation. We have further found that mixtures of fMLP and LPS behave synergistically. We believe that this synergy represents an important pathogenic mechanism during bacterial infection, but the manner by which synergy is produced is poorly understood at the molecular level. This proposal, therefore, will investigate the intracellular signaling pathways that lead to the synergistic activation of transcription factors controlling cytokine gene expression. Its main foci will be to characterize the; signaling molecules involved in the synergistic activation of leukocytes, and to analyze how a synergistic response is induced both in vitro and in mice. Our overall hypothesis is that leukocyte transcriptional activation is regulated by bacterial products operating synergistically by using multiple signaling pathways. The results may lead to a fuller understanding of host defenses against infection and of the pathogenesis of inflammatory disorders.